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CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE |
p38 Pathway Kinases as Anti-inflammatory Drug Targets
J.F. Schindler,
J.B. Monahan and
W.G. Smith*
Pfizer Global Research and Development, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
Correspondence: * corresponding author, walter.g.smith{at}pfizer.com
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ABSTRACT
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Mitogen-activated protein kinases (MAPK) are intracellular signaling molecules involved in cytokine synthesis. Several classes of mammalian MAPK have been identified, including extracellular signal-regulated kinase, c-jun N-terminal kinase, and p38 MAP kinase. p38 is a key MAPK involved in tumor necrosis factor and other cytokine production, as well as enzyme induction (cyclooxygenase-2, inducible nitric oxide synthase, and matrix metalloproteinases) and adhesion molecule expression. An understanding of the broad biologic and pathophysiological roles of p38 MAPK family members has grown significantly over the past decade, as has the complexity of the signaling network leading to their activation. Downstream substrates of MAPK include other kinases (e.g., mitogen-activated protein-kinase-activated protein kinase 2) and factors that regulate transcription, nuclear export, and mRNA stability and translation. The high-resolution crystal structure of p38 has led to the design of selective inhibitors that have good pharmacological activity. Despite the strong rationale for MAPK inhibitors in human disease, direct proof of concept in the clinic has yet to be demonstrated, with most compounds demonstrating dose-limiting adverse effects. The role of MAPK in inflammation makes them attractive targets for new therapies, and efforts are continuing to identify newer, more selective inhibitors for inflammatory diseases.
Key Words: mitogen-activated protein kinases • p38 • rheumatoid arthritis • Crohn’s disease • inflammation • drug discovery • MAPKAP kinase • intracellular signaling Abbreviations: MAPK, mitogen-activated protein kinase • ERK, extracellular signaling kinase • JNK, c-jun N-terminal kinase • COX2, cyclo-oxygenase-2 • iNOS, inducible nitric oxide synthase • MMP, matrix metalloproteinase • MAPKK, mitogen-activated protein kinase kinase • MK-2, mitogen-activated protein-kinase-activated protein kinase-2 • MAPKAP, mitogen-activated protein kinase-activated protein • MAPKKK, mitogen-activated protein kinase kinase kinase • RANKL, receptor activator of NF- B ligand • MNK, MAP kinase signal-integrating kinase • MSK, mitogen and stress-activated protein kinase • RSK, ribosomal S6 kinase • AP-1, activator protein-1 • Tpl, tumor progression locus 2 • IL, interleukin • ASK1, apoptosis signal-regulating kinase 1 • TAO, thousand-and-one amino acids • MLK3, mixed-lineage kinase 3 • TBK, transforming growth factor β activated protein kinase • TGFβ, transforming growth factor β • LPS, lipopolysaccharide • TRAF6, TNF receptor-associated factor 6 • MKK, mitogen-activated protein kinase kinase • TAK-1, TGF-beta activated protein kinase • GRK2, G-protein coupled receptor kinase-2 MKPs, MAP kinase phosphatases • PRAK, p38 regulated/activated protein kinase • IFN , interferon gamma • AREs, AU-rich elements • AUBPs, AU-rich binding proteins • TTP, tristetraprolin • hnRNP A0, heterogeneous nuclear ribonuclear protein A0 • HUR, human R antigen • PGE2, prostaglandin E2 • Hsp27, small heat-shock protein-27 • TNF-, tumor necrosis factor alpha
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INTRODUCTION
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Mitogen-activated protein kinases (MAPK) are a conserved family of enzymes that relay and propagate external stimuli, using phosphorylation cascades to generate a coordinated cellular response to the environment (Fig. 1 ). The MAPK are proline-directed serine/threonine-specific protein kinases that regulate cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis. To date, 4 distinct classes of mammalian MAPK have been identified: the extracellular signaling kinases (ERK1 and 2), the c-jun N-terminal kinases (JNK1-3), the p38 MAPK (p38, β, , and  ), and ERK5. The MAPK are activated by the dual phosphorylation of Thr and Tyr residues within a TXY activation motif by coordinated dual-specificity MAPKK, where X is Glu, Pro, and Gly in ERK, JNK, and p38 MAPK, respectively (Zhang et al., 1994; Jiang et al., 1997). MAPK are 60–70% identical to each other, yet differ in their activation loop sequences and sizes. The activation loop is adjacent to the enzyme-active site, and its phosphorylation allows the enzyme to reposition active-site residues into the optimal orientation for substrate binding and catalysis. Downstream substrates of MAPK include mitogen-activated protein-kinase-activated protein (MAPKAP) kinases and transcription factors, the phosphorylation of which, either directly or indirectly, regulates gene expression at several points, including transcription, nuclear export, and mRNA stability and translation (Kyriakis and Avruch, 2001). The cellular consequences of MAPK activation include inflammation, apoptosis, differentiation, and proliferation.
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THE ERK SIGNAL TRANSDUCTION PATHWAY
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The ERK pathway was the first MAPK pathway to be identified (Avruch, 1998). The ERK1/2 pathway is activated by numerous stimuli, including growth factors, cytokines, viral infection, and carcinogens. The signaling module consists of a three-protein kinase cascade, Raf:MEK:ERK. The ERK pathway signal is typically initiated by GTP-bound GTPase Ras, which recruits and activates Raf kinases, B-Raf, and C-Raf (the MAPKKK family). While the amino acid sequence of the Raf isoforms is highly conserved, these kinases can be distinguished by biochemical function, tissue expression, and subcellular localization, resulting in a non-redundant cellular role (Chong et al., 2003). B- and C-Raf specifically phosphorylate and activate MEK1 and MEK2, which, in turn, activate ERK1/2. ERK1/2 are responsible for the phosphorylation of the transcription factors c-fos and ELK1, and the protein kinases MNK, MSK, and RSK. The functional consequence of ERK pathway activation through engagement of growth-factor receptors includes cell-cycle regulation, proliferation, and cell survival. Oncogenic mutations found in human tumors have been linked with mutations that convert Ras to an activated oncogene (Aguirre-Ghiso et al., 2003). Oncogenic Ras induces an up-regulation of ERK1/2 signaling pathways, which causes an increase in tumor cell proliferation rates.
Although the primary role of ERK1/2-mediated signaling has long been thought to be restricted to cell growth and proliferation, it has become clear that several inflammatory processes involve ERK1/2 activation. ERK1-deficient mice were normal and fertile, but had defective thymocyte maturation and reduced expression of alpha and beta chains of the T-cell receptor (Pages et al., 1999). This indicates that ERK activity may be critical for T-cell activation, an event mediated by the AP-1 family of transcription factors. AP-1 is a pivotal transcription factor that regulates T-cell activation, cytokine production, and the production of matrix metalloproteinases, and includes members of the jun and fos families of transcription factors (Kracht and Saklatvala, 2002). Evidence suggests that inflammation-induced ERK pathway activation proceeds through a Raf-independent cascade, utilizing the signaling module Tpl2:MEK:ERK (Dumitru et al., 2000). Pharmacologic manipulation of the ERK pathway has focused on small-molecule inhibitors of MEK1/2. At least 2 classes of MEK inhibitors have been described. One class is represented by PD98059, which was discovered when the ERK pathway was used as an in vitro target (Dudley et al., 1995). In the search for inhibitors of phorbol-ester-stimulated AP-1 transactivation, a second inhibitor, U0126, was discovered (Favata et al., 1998) and was subsequently identified as working via MEK inhibition. MEK inhibitors are currently in clinical trials for oncology and are being considered for inflammation applications.
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THE JNK SIGNAL TRANSDUCTION PATHWAY
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The JNK family of enzymes are inflammation- and stress-activated MAPKs involved in the regulation of cell proliferation, survival, and apoptosis (Shaulian and Karin, 2002). Three mammalian isoforms, JNK1, JNK2, and JNK3, are encoded by distinct genes that are highly homologous, but differ in tissue expression. JNK1 and 2 are ubiquitously expressed, while JNK3 is more restricted to the central nervous system, heart, and testes (Davis, 2000). The JNKs were first identified as the enzymes responsible for the phosphorylation of the N-terminus of c-Jun, which is a component of the AP-1 transcription factor complex. The phosphorylation of c-Jun by JNK facilitates the formation of the AP-1 dimeric transcription factor c-fos:c-jun complex. The phosphorylation and activation of JNK are carried out by 2 specific MAPKK kinase enzymes, MKK4 and MKK7 (Ho et al., 2006). These 2 MAPKK are functionally distinguished, in that MKK7 is preferentially activated by cytokines such as IL-1β and TNF, while MKK4 is activated by environmental stress; however, maximal JNK activity may require both MAPKK. Inhibition of JNK-mediated AP-1 activation may prove to be a novel anti-inflammatory/immunosuppressive approach that will inhibit inducible expression of inflammatory genes in pathological conditions, including cancer, stroke, ischemic heart disease, and inflammatory disorders (Manning and Davis, 2003).
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THE p38 MAPK PATHWAY AND ITS ROLE IN INFLAMMATION
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Distinct genes encode 4 p38 MAPK in humans: p38, β, , and . Significant amino acid sequence homology is observed among the 4 isoforms, with 60%–75% overall sequence identity and > 90% identify within the kinase domains (Kumar et al., 1997). Tissue-selective expression is observed, with p38 found predominantly in skeletal muscle, p38 in the testes, pancreas, and small intestine. In contrast, p38 and β are more ubiquitously expressed.
p38 MAPK was initially discovered in a pharmacological screen when a cellular functional assay was used for the identification of compounds that modulate TNF production from a LPS-stimulated human monocytic cell line (Lee et al., 1994). This work ultimately led to the discovery of the pyridinyl-imidazole class of compounds, characterized by SB-203580 (Fig. 2). The initial mechanism for the anti-inflammatory effect of SB-203580 was thought to occur via inhibition of the cyclooxygenase and 5-lipoxygenase pathways. However, with the use of THP-1 cells and a photoaffinity radiolabeled pyridinyl-imidazole compound, 2 38-kDa proteins were identified and termed ’cytokine-suppressive anti-inflammatory drug-binding protein-1/2’, later shown to exhibit protein kinase activity and designated p38 and β (Han et al., 1994; Lee et al., 1994).
An understanding of the broad biologic and pathophysiological roles of p38 MAPK family members has grown significantly over the past decade, as has the complexity of the signaling network leading to their activation. Scientific exploration of this pathway from biological, cellular, and in vivo perspectives was largely enabled by the availability of well-behaved, selective, small-molecule inhibitors of p38 MAPK that target the and β isoforms. p38 MAPK pathway function is critical for the production and activity of multiple pro-inflammatory cytokines, including TNF, IL-1, IL-6, and IL-8, in cells such as macrophages, monocytes, synovial cells, and endothelial cells (Ajizian et al., 1999; Underwood et al., 2000) (Fig. 3). p38 MAPK is also responsible for the induction of key inflammatory enzymes such as COX2 and iNOS, the major sources of eicosanoids and nitric oxide at sites of inflammation, respectively (Dean et al., 1999). The p38 MAPK pathway regulates the expression of matrix metalloproteinases, including MMP2, MMP9, and MMP13 (Ridley et al., 1997; Underwood et al., 2000). Additionally, p38 MAPK regulates osteoclast differentiation and bone resorption through modulation of RANKL expression (Mbalaviele et al., 2006). Since most p38 MAPK inhibitors block the activity of both the and β isoforms, the role of p38β MAPK in cytokine production was determined in p38β(–/–) mice. These mice were viable, fertile, and exhibited no apparent health problems. In contrast to the pharmacological inhibition of p38 MAPK, the p38β(–/–) mice showed normal lipopolysaccharide-induced cytokine production, and in p38β(–/–) mice overexpressing TNF, joint and bowel inflammation developed (Beardmore et al., 2005). Together, these results suggest that p38 MAPK is the major isoform involved in the immune and inflammatory response. The biological roles of the and isoforms of p38 MAPK are less well-understood.
MAPK Pathway Activation
The p38 MAPK pathway is activated through stimulation of numerous classes of receptors, including G-protein-coupled receptors, cytokine receptors, Toll-like receptors, growth factor receptors, and receptors associated with environmental and genotoxic stress. Biochemical, overexpression, and molecular and genetic knockdown approaches implicate several MAPKKK (ASK1, TAO kinase 1-3, MLK3), MEKK2-4, Tpl-2, and TBK1 as upstream enzymes in the p38 MAPK pathway. These enzymes are activated by overlapping stimuli in a cell-type-dependent manner, and they target multiple signal transduction cascades in addition to p38 MAPK. The activation mechanisms for the p38 MAPK-directed MAPKKK have not been clearly elucidated, and are dependent on the specific interaction with individual receptors. Each MAPKKK may be activated by several classes of receptors, utilizing multiple signal transduction initiators. For example, ASK1 may be activated by endotoxin, inflammatory cytokines, and oxidative stress (Matsuzawa et al., 2005), while MLK3 and TAK1 can be activated by transforming growth factor-β and LPS (Lee et al., 2000; Kim et al., 2004). Transduction mechanisms following receptor stimulation leading to MAPKKK activation may include binding of small GTPases, such as Cdc42 and Rac (Du et al., 2005), interactions with MyD88 and TNF receptor-associated factor 6, Gadd45 binding, and phosphorylation by MAPKKK (Takekawa and Saito, 1998; Matsuzawa et al., 2005). The MAPKKK enzymes remain viable anti-inflammatory and immune-modulatory drug targets that could potentially induce unique biological consequences as a result of the stimuli and cell-type-specific response desired.
MKK6/MKK3 and MKK4
The direct phosphorylation and activation of MAPKs is highly regulated and mediated by the dual-specificity MAP kinase kinases (MKKs). ERK1 and 2 are specifically and uniquely phosphorylated by MEK1/2. MKK3 and 6 phosphorylate and activate p38 MAPK isoforms, while MKK4 and MKK7 activate JNK. MKK4 has also been shown to phosphorylate p38 MAPK in specific cells under the direction of select stimuli (Brancho et al., 2003). However, the physiological role of MKK4 phosphorylation of p38 MAPK has been questioned, since TNF stimulation of cells from MKK4(–/–) mice resulted in a reduction of JNK, but not p38 MAPK, activation. In addition to the classic activation pathway described above, it has been demonstrated that p38 MAPK can also be activated in T-lymphocytes following T-cell receptor engagement through phosphorylation of Y323 by the kinase ZAP70. This ZAP70-mediated phosphorylation promotes autophosphorylation of the TGY motif and subsequent activation of p38 MAPK (Salvador et al., 2005).
While the proline residue directs the phosphorylation of the MAPKs, it is insufficient to account for the high degree of MKK:MAPK selectivity observed. The specificity of the MKKs for activators, substrates, and scaffolding proteins is, in large part, regulated through docking domains that facilitate interactions with these pathway proteins (Enslen et al., 2000). These interactions may allow for independent signaling roles for these parallel pathways. The protein kinases MKK3 and MKK6 specifically activate the p38 MAPKs (Derijard et al., 1995). Among the p38 MAPK isoforms, these docking domains direct specificity such that MKK3 preferentially activates p38 and p38, while MKK6 and MKK3b activate p38, p38β, and p38 (Chang et al., 2002). TNF-stimulation of fibroblasts from MKK3(–/–), but not MKK6(–/–), mice displayed a reduction in p38 MAPK phosphorylation and activity, indicating that MKK3 plays a dominant role in the activation of p38 MAPK (Brancho et al., 2003). In contrast, many studies have documented that MKK6 is a strong activator of p38 MAPK (Han et al., 1996; Moriguchi et al., 1996a; Stein et al., 1996). For example, MKK6 is the major activator of p38 MAPK in cells exposed to osmotic stress (Moriguchi et al., 1996b).
The role of MKK3 and MKK6 in the production of cytokine and inflammatory proteins has also been examined. The disruption of the MKK3 gene results in viable, fertile mice with reduced production of IL-12 (Lu et al., 1999), IFN- (Li et al., 2005), IL-6, and IL-1β (Inoue et al., 2006). In addition, the production of COX2 (Degousee et al., 2003) and MMPs is also reduced in MKK3(–/–) cells. Thus, targeting MKK3 with small-molecule inhibitors may be beneficial in inflammatory disease.
Several regulatory and feedback mechanisms modulate the activity of the p38 pathway. The TAK-1 binding protein TAB-1 regulates the activity of the MAPKKK, TAK-1. Upon stimulation of cells with cytokines (TNF, IL-1), the TAB-1 protein is phosphorylated by p38 MAPK, resulting in a decrease in activity of TAK-1, which, in turn, down-regulates the signaling pathway through feedback inhibition. In agreement with this mechanism, inhibition of TAB-1 phosphorylation by p38 with SB203580 results in an up-regulation of TAK-1 activity (Cheung et al., 2003). Interestingly, TAB1 has also been shown to bind to p38 MAPK and induce phosphorylation and activation in a MKK3/6-independent manner in murine embryonic fibroblast cells (Kang et al., 2006), suggesting multiple roles for this protein in regulating the p38 MAPK pathway. Another p38 MAPK regulatory mechanism has been reported where G-protein-coupled receptor kinase-2 can phosphorylate p38 MAPK on a residue in its docking groove (T123), thereby preventing MKK6-induced phosphorylation and activation (Peregrin et al., 2006).
Finally, the phosphorylation state of MAPK is dynamic, regulated through upstream phosphorylation by MKKs and dephosphorylation by MAP kinase phosphatases. MAP kinase phosphatase-1 is a dual-specificity phosphatase that has been shown to dephosphorylate p38 MAPK and JNK family members preferentially (Franklin and Kraft, 1997). MAP kinase phosphatase-1 is an immediate early gene whose expression is up-regulated by stress-activated protein kinase cascades, including p38 MAPK (Hu et al., 2007).
p38 MAPK Downstream Regulation
The use of selective and potent inhibitors has facilitated the discovery of several families of p38 MAPK substrates, including transcription factors, MAPKAP kinases, and other enzymes. p38 MAPK can directly phosphorylate several transcription factors, such as myocyte-specific enhancer binding factor 2C (MEF2C), CHOP, PPAR co-activator 1, peroxisome proliferator-activated receptor , and p53 (Wang and Ron, 1996; Han et al., 1997; Barger et al., 2001). These transcription factors are involved in cellular functions such as apoptosis, gluconeogenesis, and synthesis of enzymes involved in fatty acid oxidation. p38 MAPK is also involved in the direct or indirect phosphorylation of enzyme substrates, such as cytosolic phospholipase A2 (Kramer et al., 1996), and the Cdc25 phosphatases, which are involved in the activation of cyclin-dependent protein kinase activity and cell-cycle regulation (Khaled et al., 2005; Kittipatarin et al., 2006).
The MAPKAP kinases—MK2, MK-3, and PRAK—are selectively phosphorylated by p38 MAPK, while the phosphorylation of MSK1/2, MNK1/2, and RSKb is catalyzed by both p38 MAPK and ERK (Hauge and Frodin, 2006). Activation of RSKb is thought to play a role in cell survival, although the identification of substrates has been difficult, due to the lack of specific inhibitors. MNK is involved in the phosphorylation of eukaryotic initiation factor-4E, which binds to the ’cap’ structure of mRNA and enhances protein translation (Scheper and Proud, 2002). MNK phosphorylates the mRNA binding protein hnRNP-A0, a protein that regulates mRNA stability of transcripts encoding inflammatory proteins (Buxadé et al., 2005). MSK1/2 is involved in the phosphorylation of the transcription factors CREB and ATF-1, which regulate AP-1 binding proteins (Wiggin et al., 2002). In addition, MSK1/2 can phosphorylate Histone H3, which is involved in chromatin remodeling (Dunn et al., 2005). While evidence suggests that MSK and MNK play a role in the mediation of pro-inflammatory cytokines, in vivo data with selective inhibitors and/or knockout mice are lacking.
MK-2, MK-3, and PRAK, once phosphorylated and activated by p38 MAPK, share similar substrate specificities. All of these kinases can phosphorylate the small heat-shock protein Hsp27 (Gaestel, 2006). Studies have shown that the PRAK- and MK3-deficient mice do not display any resistance to endotoxic shock or a decrease in LPS-induced cytokine production (Shi et al., 2003; Gaestel, 2006). In contrast, MK-2-deficient mice show a resistance to endotoxic shock and an impaired inflammatory response, as well as a significantly decreased production of cytokines such as TNF and IL-6 (Kotlyarov et al., 1999; Hegen et al., 2006). Thus, MK-2 is a critical p38 MAPK substrate involved with mediating pro-inflammatory responses. Like the MAPKs, MK2 requires phosphorylation on multiple amino acid residues (T25, T222, and T272) for optimal kinase activity. However, unlike the MAPKs, MK2 also has a phosphorylation site located in the hinge region (T334) between the catalytic domain and the C-terminal region (Ben-Levy et al., 1995). The C-terminal domain of non-phosphorylated MK-2 is suggested to block the binding of protein substrates. Upon phosphorylation of T334, the C-terminal domain of MK-2 moves away from the active site and allows substrate binding to occur.
Biochemical studies have established that MK2 phosphorylation is accompanied by a high-affinity docking interaction with p38 MAPK exhibiting a binding constant of 2.5 nM for the non-phosphorylated forms of both enzymes. The docking site of MK-2 is characterized by a cluster of positively charged amino acids (370IKIKKIEDASNPLLLKRRKKARALEAAALAH400) located within the C-terminal domain of the enzyme. The C-terminal domain of MK-2 binds to the common docking domain of p38, which is composed of an acidic patch of amino acids (Asp316, Asp319), adjacent to, but distinct from, the docking groove where MEF2A and MKK3b bind (Chang et al., 2002). This interaction between p38 MAPK and MK-2 has been structurally confirmed in the crystal structure of the non-phosphorylated heterodimeric complex of p38:MK-2 (ter Haar et al., 2007; White et al., 2007).
The high-affinity interaction between p38 MAPK and MK-2 has also been confirmed in cells. Studies have suggested that p38 MAPK and MK-2 exist as a pre-formed complex, primarily located within the nucleus (Ben-Levy et al., 1998). The localization of MK-2 is governed by a nuclear localization signal found in the C-terminal domain (373-389 KKIEDASNPLLLKRRKK; key residues in bold) (Gaestel, 2006). Upon cellular stimulation, p38 MAPK is activated and, in turn, phosphorylates MK2, leading to the translocation of the p38:MK-2 complex to the cytoplasm (Fig. 4). MK-2 contains a nuclear export signal in the C-terminal domain (328MTSALATMRV351), which is repositioned upon phosphorylation by p38 MAPK, allowing for translocation from the nucleus to the cytoplasm, where both kinases can phosphorylate their respective substrates (Engel et al., 1998). The p38 MAPK inhibitor SB203580 can block the export of p38 and MK-2 from the nucleus, suggesting that the phosphorylation of MK-2 is the key to the translocation mechanism (Ben-Levy et al., 1998).
Inflammatory Gene Regulation by p38 MAPK/MK2
The p38 MAPK-mediated regulation of inflammatory gene transcription and translation is, in major part, MK2-dependent and involves multiple mechanisms (Fig. 4 ). mRNA levels of cytokines IL6 and INF, as well as COX2, were decreased in LPS-stimulated MK-2(–/–) mice, suggesting a role for MK-2 in the stabilization of mRNAs. In contrast, TNF is regulated in these mice at the translational level, in that protein levels are significantly reduced without concomitant changes in mRNA. The mechanism of this p38 MAPK:MK2 regulation of inflammatory protein production is not clear, but may involve phosphorylation of mRNA binding proteins. mRNA transcripts that rapidly turn over—such as those encoding COX2, INF, IL-1, IL-2, IL-3, IL-6, and IL-8 proteins—frequently contain AU-rich elements (AREs) in the 3' untranslated region. Several AU-rich binding proteins (AUBPs) that regulate message stability have been identified, including TTP, hnRNP A0, and HuR. Since the activation of p38 MAPK and MK-2 leads to the stabilization of ARE-containing transcripts, the AUBPs are likely targets for the p38 MAPK/MK2 pathway.
One example of this regulation is the zinc finger-ARE binding protein TTP, a downstream substrate p38 MAPK:MK-2 (Hitti et al., 2006). TTP binds to the ARE motifs of TNF mRNAs and targets them for degradation. The phosphorylation of TTP by MK-2 facilitates the dissociation of TTP from the ARE motif, allowing for the biosynthesis of TNF. The role of TTP has been shown with TTP(–/–) mice, which developed severe inflammatory symptoms due to the overproduction of TNF (Taylor et al., 1996). Several reports have also implicated MK-2 in the regulation of hnRNP A0 and HUR (Rousseau et al., 2002; Gaestel, 2006), although a specific link between MK2-induced phosphorylation and function is lacking.
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p38 MAPK STRUCTURE AND FUNCTION
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The structure of the unphosphorylated apoform of p38 MAPK has been determined by x-ray crystallographic analysis (Wilson et al., 1996). p38 MAPK, like most protein kinases, is characterized by 2 domains separated by a deep channel where potential substrates may bind. The N-terminal domain creates a binding pocket for ATP, and the C-terminal domain contains the catalytic residues involved in the transfer of the gamma phosphate to the protein substrate. The orientation of the 2 domains of non-phosphorylated p38 MAPK is different from the crystal structure described for cAMP, which is believed to represent a bioactive conformation of protein kinases. When compared with cAMP, the p38 MAPK domains are distorted, so that essential catalytic residues are not in the optimal position to promote catalysis, suggesting that the 2 domains need to reposition for efficient phosphate transfer.
The high resolution of the apo-non-phosphorylated form of p38 MAPK led to the design of the ATP competitive inhibitor SC80036, which replaced the pyridinyl-imidazole central scaffold found in SB203580 with a pyridinyl-pyrazole group (Fig. 2). The pyridinyl-imidazoles were instrumental in the discovery of p38 MAPK as a target, but did not have the physical properties for oral drug delivery, and faced several adverse effects associated with off-target pharmacology (Adams et al., 2001). The primary role of the central imidazole is to serve as the scaffold to orient the vicinal aryl pyridinyl ring. The substitution of the central imidazole with pyrrole, pyrazoline, pyrazole, or 7-azindole provides potent inhibitors of p38 MAPK. In some cases, these substitutions lead to improved potency and minimized liabilities, which were observed for the pyridinyl-imidazole class.
The crystal structure of the p38 MAPK/SC80036 complex confirms the binding of SC80036 to the ATP pocket (Fig. 5a). The p38 MAPK/SC80036 crystal structure explains the potency and selectivity observed with this class of p38 MAPK inhibitors (Burnette et al., submitted to J Pharmacol Exp Ther). One edge of the pyrazole moiety is stabilized by interactions with the flexible glycine flap (residues 33–39, containing the consensus motif, Gly-X-Gly-X-X-Gly), while the central pyrazole formed 2 water-mediated interactions with Lys53 and Asp168. The pyrimidine ring forms a hydrogen bond with the peptide nitrogen of Met109. Significantly, the 4-chlorophenyl group was bound in the p38 lipophilic pocket, which is the basis for the high degree of selectivity observed with this and other inhibitors. T106 is located at the entrance to the deep lipophilic pocket of p38 MAPK and p38β MAPK, and acts as a "gatekeeper", allowing for efficient binding of this class of inhibitor to these p38 MAPK isoforms. In 80% of protein kinases, including p38 MAPK and p38 MAPK, the gatekeeper residue is a bulkier amino acid such as methionine, which does not allow inhibitors to gain access to the hydrophobic pocket.
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Figure 5. Crystal structure of the ATP binding pocket of: (a) p38a/SC80036 complex, (b) p38a/VX-745 complex, (c) p38a/BIRB-796 complex, and (d) MK2/compound 23 complex.
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The selectivity of p38 MAPK inhibitors is due to the access that these inhibitors have to the hydrophobic pocket. While the first-generation inhibitors had reasonable selectivity against several kinases, these compounds inhibited other kinases, including JNK isoforms and, to a lesser extent, ERK. Recently, the p38 MAPK inhibitor VX745 (Fig. 2) has been described as having greater selectivity for p38 MAPK compared with first-generation inhibitors (Fitzgerald et al., 2003). Structural studies revealed that the hinge region of p38 MAPK undergoes a backbone flip at Gly110, to provide 2 backbone amide groups (Met109 and Gly110) for hydrogen binding to the carbonyl moiety of VX745 (Fig. 5b). This flip is much more energetically favorable for p38, β, and MAPKs, where residue 110 is Gly, compared with other kinases, such as JNK and ERK, where Gly110 is replaced with a bulkier amino acid residue. A homology search across the human kinome revealed that very few protein kinases contain Gly in this key position and share the ability to undergo the backbone flip, which gives rise to an enhanced p38 MAPK selectivity.
The third class of p38 MAPK inhibitors can be characterized by BIRB-796 (Fig. 2), with a unique binding mode (Pargellis et al., 2002). Crystallographic studies revealed that the tertiary-butyl group on the pyrazole core binds in a hydrophobic pocket that other p38 MAPK inhibitors do not access (Fig. 5c). Access to this hydrophobic pocket is gained by a 10 Å displacement of the F169 residue within the DFG motif by the tertiary-butyl group. In contrast to the first- and second-generation p38 MAPK inhibitors, the binding of BIRB-796 causes a disruption in the activation loop of p38 MAPK, and blocks the phosphorylation of p38 MAPK by MKK6 (Sullivan et al., 2005), locking p38 MAPK into a non-active conformation. In addition to this unique binding property, BIRB-796 displays a very slow dissociation rate from the enzyme (T1/2 > 24 hr), compared with the very rapid dissociation observed with first- and second-generation inhibitors (T1/2 < 1 min).
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p38 INHIBITOR PHARMACOLOGY
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The observation that most ATP-competitive p38 MAPK inhibitors bind with similar affinity to both the activated and non-activated kinases, while MgATP strongly prefers the phosphorylated, active form of the enzyme, suggests that these inhibitors will maintain enzyme potency in cellular systems. This hypothesis is in contrast to many protein kinase inhibitors that demonstrate a significant decrease in potency when cellular and isolated enzyme activities are compared, most likely due to competition with high cellular ATP. The potential for direct translation of isolated enzyme potency to cellular and in vivo potency adds support to p38 MAP kinase as a ’druggable’ kinase target.
The binding affinity of SC80036 to p38 MAPK has been determined in biophysical studies. SC80036 has a Ki = 62 nM for the non-phosphorylated enzyme, which is similar to the Ki value determined with phosphorylated p38 MAPK (Ki 35 nM) (Schindler and Monahan, unpublished observations). In contrast, MgATP does not bind the non-phosphorylated form of p38 MAPK (Ki > 5 mM), consistent with the distorted ATP binding domain observed in the crystal structure of non-phosphorylated p38 MAPK.
SC80036 antagonizes the cellular production of key inflammatory mediators, including TNF, PGE2, IL-1 and IL-6, following endotoxin stimulation of human monocytic cells and whole blood with potency similar to that of enzyme inhibition in the biochemical assay. Further evidence in support of the direct enzyme-to-cell translation was generated with cellular biomarkers of p38 MAPK phosphorylation activity. Cellular activity was determined by the measurement of activity of the downstream substrate MK2 or the phosphorylation of HSP27, an MK2 substrate (Burnette et al., submitted to J Pharmacol Exp Ther). Results in U937 cells and whole blood, where p38 MAPK cellular activity was inhibited by SC80036, with potency comparable with inhibition of inflammatory biomarkers in the same cell, are shown in Fig. 6.
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Figure 6. Ex vivo correlation of inhibition of p38 MAPK activity with that of inflammatory cytokine release by SC80036 in (A) U937 cells or (B) human whole blood (Burnette et al., submitted to J Pharmacol Exp Ther).
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The effect of SC79659, an analog of SC80036, on TNF and p38 MAPK target (MK2 activity) biomarker measurements was extended in vivo following acute endotoxemia in rats, where TNF suppression mirrored MK2 activity (Fig. 7). This approach was further extended to human clinical studies, where participants were given low-dose endotoxin following SC80036 administration, and blood cytokine and p38 MAPK activity were measured (Burnette et al., submitted to J Pharmacol Exp Ther). Interestingly, TNF and p38 MAPK activity levels were decreased in parallel, in a dose- and concentration-dependent manner (Fig. 8).
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Figure 7. Effect of SC79659 on LPS-induced inflammatory cytokine production in rodents (Burnette et al., submitted to J Pharmacol Exp Ther).
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Figure 8. In vivo inhibition of TNF- release by SC80036 in a human experimental endotoxemia clinical trial and correlation with inhibition of p38 MAPK activity (Burnette et al., submitted to J Pharmacol Exp Ther).
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SC80036 demonstrated significant efficacy in arthritis and pain models, including rat streptococcal cell wall arthritis, mouse collagen-induced arthritis, and rat carra-geenan-induced paw inflammation and pain models. Overall, these pre-clinical and clinical studies support the direct translation of p38 MAPK inhibition across biochemical, cellular, and in vivo models, and demonstrate that selective pharmacological modulation of p38 MAPK is sufficient to suppress arthritis and bone erosion in animal models of disease with efficacy equal to that of anti-TNF biologics and steroids.
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MK-2 STRUCTURE, FUNCTION, AND PHARMACOLOGY
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The crystal structure of non-phosphorylated MK-2 has been solved (Underwood et al., 2003). MK-2 is bi-lobal, with an N-terminal domain and a C-terminal domain that make up the active site. In contrast to p38 MAPK, the crystal structure of non-phosphorylated MK-2 reveals that the residues involved in the binding of ATP are in the optimal catalytic positions. Consistent with this structural information, it was observed that non-phosphorylated MK-2 is capable of binding MgATP with reasonable affinity (30 µM), in contrast to p38 MAPK, which displays essentially no binding to MgATP (Schindler and Monahan, unpublished observations). However, non-phosphorylated MK-2 displays very low activity when compared with the phosphorylated form of MK-2. The low catalytic activity of non-phosphorylated MK-2 is due to the autoinhibitory C-terminal domain, which blocks the binding of protein substrates. The phosphorylation of MK-2 by p38 MAPK triggers the displacement of the auto-inhibitory domain of MK-2, thereby permitting the binding of protein substrates. This activation mechanism was confirmed in binding studies, which demonstrated that only the phosphorylated form of MK-2 will bind to an inhibitor peptide substrate (Schindler et al., 2002).
Potent and selective MK-2 inhibitors based on a pyrrolepyridine scaffold have been disclosed (Fig. 2) (Anderson et al., 2007). The crystal structure of Compound 23 bound to the nucleotide binding site of non-phosphorylated MK-2 is shown in Fig. 5d. The compound is a potent ATP-competitive inhibitor of MK-2 (Ki = 0.126 µM), which is borne out by crystallographic data. Additional work has also shown that Compound 23 binds to non-phosphorylated MK-2 with similar affinity (Ki = 0.145 µM) (Schindler and Monahan, unpublished observations). Oral administration of Compound 23 to rats resulted in an 87% inhibition of LPS-induced TNF production. Progress in the identification of potent and selective MK2 inhibitors with cellular activity has been very slow, which may be a reflection of the facts that: (1) the nucleotide-binding pocket of MK-2 is flat, with limited hydrophobic pockets available for interaction with inhibitors; and (2) the binding competition with inhibitors and ATP in both the non-activated and activated enzymes may result in significantly lower cellular vs. isolated enzyme potency, due to the high concentrations of ATP in the cell.
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p38 MAPK INHIBITORS AS THERAPEUTIC AGENTS
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p38 MAPK’s role in the regulation of inflammatory cytokines and enzymes responsible for inflammation—like COX2, iNOS, and MMPs—makes it an attractive drug target. Several p38 MAPK inhibitors have progressed to testing in clinical trials (Table). Some of these candidates have failed, for safety or other reasons, but several have reported clinical data (Dominguez et al., 2005; Goldstein and Gabriel, 2005; Lee and Dominguez, 2005). The potential for MAPK inhibitors in the dental field is specifically addressed in the accompanying manuscript in this issue (Patil and Kirkwood, 2007).
Rheumatoid Arthritis (RA)
Rheumatoid arthritis (RA) affects about 1.0% of the adult population worldwide and is characterized by polyarticular synovitis with a prominent immunological, inflammatory, and mesenchymal tissue reaction in the synovium, causing pain, swelling, stiffness, and bone and cartilage destruction (Calvo and Alarcon, 2000). The infiltration of cells such as macrophages and T-cells into the synovial lining is prominent, as are the proliferation and expansion of fibroblasts. RA synovial fluid is enriched with neutrophils, macrophages, T-cells, and dendritic cells. Many of these cells have an activated phenotype, expressing high levels of human leukocyte antigen class II antigens and adhesion molecules, and produce most of the known cytokines and chemokines. Anti-cytokine biotherapeutic approaches—such as etanercept (soluble TNF receptor), infliximab, and adalimumab (anti-human TNF monoclonal antibodies)—have dramatically changed the treatment options for persons with RA, not only reducing signs and symptoms, but also providing disease-modifying effects, as evidenced by the lack of progression of radiologic bone loss (Kievit et al., 2007).
The success of the anti-TNF biotherapeutics has led to the extensive evaluation of p38 MAPK inhibitors in pre-clinical models of arthritis. However, convincing proof of concept from clinical trials has not been achieved. VX-745 was reported to achieve an American College of Rheumatology (ACR) 20 response in 43% of treated persons, compared with 17% for placebo (Weisman et al., 2002). In this same study, it was reported that there was an exposure-dependent reduction in CRP and IL-6. VX-702, another selective p38 inhibitor being developed by Vertex (Cambridge, MA, USA) and Kissei (Matsumoto City, Nagano, Japan), has been evaluated in a 12-week study of 315 persons with RA in Eastern and Central Europe. The company reported that the drug was well-tolerated at the doses tested (5 and 10 mg, QD). At 12 weeks, 40% and 38% of persons taking the high and mid doses, respectively, achieved an ACR-20 score, compared with 30% on placebo. Side-effects reported included rash, infection, and gastrointestinal intolerance. Other p38 MAPK inhibitors have been studied in RA or are currently in clinical studies. SCIO-469 has been reported to have been tested in a 12-week RA study, where it elicited an ACR-20 score in 53% of persons, compared with 23% with placebo (unpublished data, analysts’ report). Currently under way is a 12-week Phase II study of PH-797804 (Pfizer) in individuals with active RA. Paramount to an understanding of the role of p38 MAPK inhibitors in RA is an ability to track exposure-driven effects on a target biomarker. Studies with VX-745 and VX-702 may have been limited by side-effects, and it is not known if additional benefit could be derived from increased exposure.
Crohn’s Disease
Crohn’s disease is characterized by a chronic inflammation in the gastrointestinal tract, linked to a mucosal immune response to gut microflora. There are increased tissue levels of inflammatory cytokines (IL-1, IL-6, TNF), and anti-TNF biologics (etanercept, infliximab, adalimumab) are clinically efficacious (Danese et al., 2006). To date, 2 studies have been reported with small-molecule MAPK inhibitors. CNI-1493, a non-selective p38 and JNK inhibitor, was tested in 12 persons with severe Crohn’s disease by IV administration for 12 days (Hommes et al., 2002). A statistically significant improvement in the Crohn’s Disease Activity Index was seen after 4 and 8 weeks, with no serious adverse events. BIRB-796 was tested in an 8-week study of 284 persons with moderate to severe Crohn’s Disease, and failed to show a clinical benefit (Schreiber et al., 2006). The reason for the lack of efficacy is not known, but could be related to lower-than-expected exposures to the compound.
Pain
Inflammatory mediators (e.g., prostaglandins and nitric oxide) and cytokines (e.g., TNF and IL-1) are known to contribute to peripheral and central sensitization and to modulate acute, chronic, and neuropathic pain (Watkins et al., 2005). MAPKs have been shown to be activated in primary sensory neurons and glia in the spinal chord (Ji, 2004), and therefore the role of p38 MAPK inhibitors in pain states is being explored. In a model of dental pain, SCIO-469 has been studied compared with ibuprofen, with the time to rescue pain medication as the efficacy endpoint. In this study of 263 individuals, 150 or 300 mg of SCIO-469, administered prior to oral surgery, extended the time to rescue pain medication from 4.1 hours in placebo to 6.1 and 5.7 hrs in the low and high doses, respectively. The most significant response (8.1 hrs) was achieved by the administration of 210 mg prior to and another 90 mg at the time of surgery, suggesting an analgesic effect of the compound. Currently under way is a double-blind, placebo-controlled study of orally administered SB-681323 (7.5 mg, BID) in individuals with neuropathic pain following nerve trauma.
Acute Coronary Syndrome (ACS)
Persons with acute coronary syndrome (ACS) are at high risk of death and/or the recurrence of serious cardiovascular events. ACS is due to the disruption of atherosclerotic plaque, which leads to platelet adhesion and aggregation, followed by intra-coronary clot formation. Persons with ACS have increased levels of C-reactive protein, a systemic marker of inflammation, and higher levels of activated monocyte/macrophages, which lead to elevated levels of IL-6 (van Haelst et al., 2004). VX-702 was administered prior to and after angioplasty in persons with elevated CRP (Monhanial et al., 2007, unpublished observations). The compound was well-tolerated and decreased CRP, but clinical outcomes were similar to those with a placebo. SB-681323 is currently undergoing a 28-day randomized, double-blind, placebo-controlled study in individuals with coronary heart disease undergoing percutaneous coronary intervention.
Other Diseases
Chronic obstructive pulmonary disease (COPD) is associated with an inflammation of the airways and lung parenchyma, characterized by activated alveolar macrophages and increased numbers of activated cytotoxic (CD8+) T-cells and neutrophils. Macrophages may be activated by cigarette smoke to release inflammatory mediators, including TNF, IL-8 and other CXC chemokines, monocyte chemotactic peptide-1, leukotriene B4, and reactive oxygen species, providing a cellular mechanism that links smoking with lung inflammation. The trafficking and activation of neutrophils result in the release of numerous inflammatory mediators, such as proteases (MMPs and neutrophil elastase), which contribute to the progressive fibrosis, airway narrowing, and destruction of the lung parenchyma that lead to an accelerated decline in airway function. In a pre-clinical model of COPD, SB-239063 reduced neutrophil infiltration after inhaled endotoxin and the concentrations of IL-8 and IL-6 and matrix metalloproteinases-9 in the broncho-alveolar lavage fluid of rats (Underwood et al., 2000). Of interest would be an exploration of the potential of p38 MAPK inhibitors to work via the inhaled route, which may allow for exposure to the target tissue while limiting systemic drug concentrations.
BIRB-796 (5–30 mg, BID) has been explored in a 4-week trial of plaque-type psoriasis. The primary endpoint was epidermal thickness assessed on skin biopsies on days 0, 7, and 28. The Psoriasis Area and Severity Index, T-cell infiltrate, keratinocyte proliferation, and cytokines were secondary endpoints. A positive result was achieved for the primary endpoint, but this was not associated with a clinically detectable improvement in lesions. Again, the reason for the lack of clinical efficacy may be related to the dose-related inhibition of p38 MAPK, since higher doses (50 mg) were needed in previous studies before an affect on ex vivo neutrophil activation could be seen (Kreuger et al., unpublished observations).
Multiple myeloma is a B-cell malignancy with plasma cells phenotypically expressing CD38+, CD56+, and CD138+. Overproduction of IL-6 (a plasma cell growth factor), TNF, and IL-1 can be seen. In an in vitro study, SCIO-469 was investigated as a potential anticancer agent (Nikas and Drosos, 2004). The compound was tested as both a single agent, and in combination with proteasome inhibitor. In multiple myeloma cells, p38 MAPK activation and cytokine production were inhibited by SCIO-469, although there was little effect on the viability of the tumor cells. However, in combination with proteasome inhibitors, SCIO-469 enhanced the reduction in multiple myeloma cell proliferation and increased proteasome inhibitor-induced apoptosis.
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CONCLUSIONS
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Signal transduction initiated by receptor activation triggers a complex set of cascading networks involving significant crosstalk, intracellular trafficking, scaffold modules, and feedback loops. The net cellular response is dependent on signal strength, amplification, and duration, protein expression levels, and the numbers and types of concurrent extracellular signals. As these complex systems become more clearly understood, in both normal and pathological states, key intervention points are being identified which may allow for the discovery of efficacious and safe drug candidates.
MAPKs such as p38 MAPK are not only involved in the regulation of cytokine expression, but also maintain a significant role in cellular-adaptive responses, such as apoptosis cell-cycle regulation and proliferation, and involvement in development and differentiation. Downstream targets such as MK-2 may offer an alternative approach to regulating many of the inflammatory mediators under the control of p38. However, the ’drugabilty’ of this target has proven to be challenging. Upstream targets like MKK3/MKK6 may be viable drug targets, but we are unaware of anyone making significant progress in this area. Finally, JNK enzymes play a role in regulating many of the same inflammatory genes. But progress in identifying safe and effective drugs has been slow, most likely due to the need to have selectivity across the JNK family. Pre-clinical studies with p38 inhibitors have repeatedly demonstrated significant efficacy in many disease models, including chronic inflammation, arthritis, pain, and airway disease. A clinical utility and therapeutic index in humans is still to be determined, and most compounds have run into dose-limiting toxicities that have limited development. The most common target organs are the liver, skin, and the CNS. Whether these side-effects are mechanism-related or due to secondary pharmacology of the compounds is not known. Alternative strategies are being pursued to overcome these issues, including the identification of newer classes of compounds with greater specificity, and a deeper knowledge of the other targets in the pathway that could lead to novel treatments for inflammatory diseases
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ACKNOWLEDGMENTS
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The authors thank the following individuals for their contributions to the p38 MAPK project at Pfizer: Barry Burnette, Rajesh Devraj, Ravi Kurumbail, Gail Jungbluth, and Shaun Selness. JS, JM, and WS are all employees of Pfizer Global Research and Development and own stock in the company.
Received for publication May 4, 2007.
Revision received July 5, 2007.
Accepted for publication July 6, 2007.
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Journal of Dental Research, Vol. 86, No. 9,
800-811 (2007)
DOI: 10.1177/154405910708600902
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TNF-{alpha} Promotes an Odontoblastic Phenotype in Dental Pulp Cells
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ABSTRACT
INTRODUCTION
